PROJECT 1: HDL AND CELLULAR CHOLESTEROL METABOLISM This project will focus on the cellular and serum factors that affect cholesterol transport between cells and high density lipoprotein (HDL)/ apoproteins. Several cell types will be used but study of macrophages will be emphasized. In Specific Aim 1 cellular factors affecting cholesterol efflux will be studied. The hypothesis to be tested is that the expression level of transporters and the contribution of aqueous diffusion in cholesterol donor cells together with the concentration/distribution of acceptor HDL particles optimal for these pathways determine efflux efficiency. We will quantify the contributions of aqueous diffusion and the SR-BI, ABCA1 and ABCG1 transporters to efflux from cells exposed to serum, isolated lipoproteins and reconstituted HDL. Protocols using both genetic manipulation and pharmacological inhibitors to measure pathway contributions to total efflux that were developed during the current grant cycle will be employed. We will also study if removal of the cholesterol produced by cholesteryl ester (CE) hydrolysis in macrophage foam cells occurs via specific efflux pathways and how oxidation of CE by lipoxygenases affects clearance. Serum factors that affect cholesterol flux will be considered in Specific Aim 2. These experiments will test the hypothesis that HDL remodeling by LCAT, CETP and PLTP alters the relative contribution of different pathways. We predict that there will be a reciprocal relationship between the effects of serum factors that increase small/lipid poor HDL and serum factors that increase large phospholipid-rich HDL.Specific Aim 3 is designed to demonstrate that the efflux capacity of human serum HDL depends on HDL "quality" and not just total HDL cholesterol. We will use the serum HDL fraction from specimens having similar total HDL-C but different efflux capacities to understand how macrophage cholesterol efflux is affected by HDL composition. In Specific Aim 4 we will use an assay we developed to simultaneously measure efflux and influx of cholesterol when macrophages are incubated with serum or lipoproteins. These assays will yield values for influx, efflux and net flux when macrophages are exposed to a variety of cholesterol acceptors. The studies proposed in Project 1 arise from close collaborations established in the past with Projects 2 and 3, and will continue to be well integrated with studies proposed in those projects. The accumulation of cholesterol-loaded macrophages in the arterial wall is an early event in formation of atherosclerotic plaques. We know this process is reduced by the presence of high circulating HDL levels but do not understand why. These studies will add to our understanding of HDL function. Children's Hospital of Philadelphia Pediatrics/GI-Hepatology-Nutrition Joseph Stokes Jr. Research Institute 3615 Civic Center Boulevard Philadelphia, PA 19104-4318 PHS 398 (Rev. 04/06) Form Page 2 81 Principal Investigator / Program Director (Last, First. Middle): Project 1 KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information Start with Principal Investigator(s). List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Rothblat, George H. Children's Hospital of GROTHBLAT Research Prof, of Pediatrics Philadelphia de la Llera-Moya, Margarita Children's Hospital of Research Assistant Prof, of MDMOYA Philadelphia Pediatrics Phillips. Michael C. in the format shown below. Role on Project Project Leader Co-Investigator Phillips, Michael C. Research Prof, of Pediatrics Weibel, Ginny Research Scientist OTHER SIGNIFICANT CONTRIBUTORS Name Asztalos, Bela F. Ghosh, Shobha Jessup, Wendy Children's Hospital of PHILLIPSMC Philadelphia Children's Hospital of WEIBELG Philadelphia Co-Investigator Co-Project Leader Organization Role on Project Tufts University Collaborator Virginia Commonwealth University Collaborator University of New South Wales Collaborator Human Embryonic Stem Cells ^ No O Yes If the proposed project Involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://Stemcells.nih.gov/registry/index.aSP. Usecontinuation pages as needed. If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line PHS 398 (Rev. 04/06) Form Page 2-continued 82 Principal Investigator/ Program Director (Last, First, Middle): PROJECT 1 DETAILED BUDGET FOR INITIAL BUDGET PERIOD DIRECT COSTS ONLY PERSONNEL (Applicant organization only) Months Devoted to Proiect ROLE ON Cal. Acad. Summer INST.BASE NAME PROJECT Mnths Mnths Mnths SALARY Principal Rothblat, George 6.00 186,600 Invpstinatnr Weibel, Ginny Co PI 5.40 85,697 Phillips, Michael Co-Investigator 1.20 186,600 De la Llera-Moya,M. Co-Investigator 3.60 96,455 Sr. Res. Nguyen, Vinh 6.00 48,620 Technician Research Hayes, Sara 3.00 30,900 Technician SUBTOTALS ^w CONSULTANT COSTS EQUIPMENT (Itemize) Phillips. Michael C. FROM THROUGH 12/01/08 11/30/09 DOLLAR AMOUNT REQUESTED (omit cents) SALARY FRINGE REQUESTED BENEFITS TOTAL 93,300 27,897 121,197 38,564 12,340 50,904 18,660 5,579 24,239 28,937 8,652 37,589 24,310 7,779 32,089 7,725 2,472 10,197 211,496 64,719 276,215| SUPPLIES (Itemize by category) Radioisotopes 6,200 Commercial Enzymatic Kit 3,300 TLC Plates 1,500 Small Equipment 2,100 Enzyme - Inhibitors 1,200 Chemicals/General Lab Supplies 16,000 TRAVEL Scientific Meeting 1,500 PATIENT CARE COSTS INPATIENT OUTPATIENT ALTERATIONS AND RENOVATIONS (Itemize by category) OTHER EXPENSES (Itemize by category) Publication costs 1 ,000 Federal Express 528 CONSORTIUM/CONTRACTUAL COSTS SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET CONSORTIUM/CONTRACTUAL COSTS GLC Supplies 3,500 Scintillation Fluid/Supplies 2,800 Organic Solvents 1,800 Miscellaneous Glassware 1,300 Biochemical Reagents 1,600 41,300 1,500 1,528 DIRECT COSTS PERIOD (Item 7a, Face Page] $ 32 0 543 FACILITIES AND ADMINISTRATIVE COSTS TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD $ 320,543 PHS 398 (Rev. 04/06) Form Page 4 83